Human Erythrocyte Cell Membrane Research Articles

Alterations in membrane stability after in vitro exposure of human erythrocytes to 2.41 GHz electromagnetic field

The growing use of wireless communication devices has been significantly increasing the level of high frequency electromagnetic fields (EMFs) in the environment, which raises a concern for possible deleterious effects on living organisms. Long lasting exposure to low-intensity EMFs can cause effects on the molecular and cellular level, and a number of morphological and physiological changes. The aim of this work was to investigate the effects of 2.41 GHz EMF emitted by wireless communication systems on human erythrocytes after in vitro irradiation. The amount of the hemoglobin released from the cells was measured as an indicator for membrane destabilization. Effects of different exposure times (20 min or 4 h) and time elapsed after exposure to 2.41 GHz pulsed or continuous EMFs with different intensities, emitted from a textile (0.213–0.238 V/m) or a dipole (5, 20, 40 and 180 V/m) antenna, were investigated. The obtained results showed that the low intensity EMF had no significant effect on the hemoglobin release from irradiated cells; even a slight tendency for membrane stabilization was noticed 3–4 hours after the end of 20-min exposure to 0.213–0.238 V/m, 2.41 GHz EMF. There was no difference in the effects of continuous and pulsed EMFs. Increased hemoglobin release was observed only during the 4-hour exposure to 180 V/m, 2.41 GHz continuous EMF. Under these conditions, the temperature of the cell suspension had been rising, so we compared the results obtained under EMF with the effects of conventional heating. Moreover, after 1-hour exposure to 180 V/m the released hemoglobin level was a bit higher than the control one but the difference disappears within an hour after terminating the irradiation. In conclusion, the in vitro exposure to 2.41 GHz EMF emitted by wireless communication devices with power density below the reference level for population exposure does not change the stability of the cell membrane of human erythrocytes.

Unidirectional fluxes of monovalent ions in human erythrocytes compared with lymphoid U937 cells: Transient processes after stopping the sodium pump and in response to osmotic challenge.

Recently, we have developed software that allows, using a minimum of required experimental data, to find the characteristics of ion homeostasis and a list of all unidirectional fluxes of monovalent ions through the main pathways in the cell membrane both in a balanced state and during the transient processes. Our approach has been successfully validated in human proliferating lymphoid U937 cells during transient processes after stopping the Na/K pump by ouabain and for staurosporine-induced apoptosis. In present study, we used this approach to find the characteristics of ion homeostasis and the monovalent ion fluxes through the cell membrane of human erythrocytes in a resting state and during the transient processes after stopping the Na/K pump with ouabain and in response to osmotic challenge. Due to their physiological significance, erythrocytes remain the object of numerous studies, both experimental and computational methods. Calculations showed that, under physiological conditions, the K+ fluxes through electrodiffusion channels in the entire erythrocyte ion balance is small compared to the fluxes through the Na/K pump and cation-chloride cotransporters. The proposed computer program well predicts the dynamics of the erythrocyte ion balance disorders after stopping the Na/K pump with ouabain. In full accordance with predictions, transient processes in human erythrocytes are much slower than in proliferating cells such as lymphoid U937 cells. Comparison of real changes in the distribution of monovalent ions under osmotic challenge with the calculated ones indicates a change in the parameters of the ion transport pathways through the plasma membrane of erythrocytes in this case. The proposed approach may be useful in studying the mechanisms of various erythrocyte dysfunctions.

Stimulating Effect of Terfenadine on Erythrocyte Cell Membrane Scrambling

Background/Aims: The antihistaminic drug Terfenadine may trigger apoptosis of tumor cells, an effect unrelated to its effect on histamine receptors. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling triggering eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, and ceramide. The present study explored, whether Terfenadine is capable to trigger eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence, and ceramide abundance at the human erythrocyte surface utilizing specific antibodies. Hemolysis was quantified from haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Terfenadine (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells and triggered hemolysis without significantly modifying the average forward scatter. Terfenadine (7.5 µM) significantly increased Fluo3-fluorescence, but did not significantly modify DCF fluorescence or ceramide abundance. The effect of Terfenadine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca²⁺. Exposure of human erythrocytes to Ca²⁺ ionophore ionomycin (1 µM, 15 min) triggered annexin-V-binding, an effect augmented by Terfenadine pretreatment (10 µM, 48 hours). Conclusions: Terfenadine triggers phospholipid scrambling of the human erythrocyte cell membrane, an effect in part due to entry of extracellular Ca²⁺ and in part due to sensitizing human erythrocyte cell membrane scrambling to Ca²⁺.

Stimulating Effect of Manumycin A on Suicidal Erythrocyte Death

Background/Aims: The streptomycete derived farnesyltransferase inhibitor Manumycin A triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. The present study explored whether Manumycin A could similarly stimulate eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include Ca²⁺ entry as well as activation of staurosporine sensitive protein kinase C and SB203580 sensitive p38 kinase. The present study explored, whether Manumycin A induces eryptosis and, if so, to shed some light on the mechanisms involved. Methods: Phosphatidylserine abundance at the human erythrocyte surface was estimated from annexin-V-binding, cell volume from forward scatter, and hemolysis from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Manumycin A (≥ 5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly incrased hemolysis. The effect of Manumycin A on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺, by addition of staurosporine (1 µM) and by addition of SB203580 (2 µM). Conclusions: Manumycin A triggers hemolysis, cell shrinkage and phospholipid scrambling of the human erythrocyte cell membrane. The effect on cell membrane scrambling was in part but not fully dependent on entry of extracellular Ca²⁺, as well as activity of staurosporine and SB203580 sensitive kinases.

Enhanced Eryptosis Following Exposure to Carnosic Acid

Background/Aims: The phenolic abietane diterpene component of rosemary and sage, carnosic acid, may either induce or inhibit apoptosis of nucleated cells. The mechanisms involved in the effects of carnosic acid include altered mitochondrial function and gene expression. Human erythrocytes lack mitochondria and nuclei but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the stimulation of eryptosis include oxidative stress, increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), and ceramide formation. The present study explored, whether and how carnosic acid induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to carnosic acid significantly increased the percentage of annexin-V-binding cells (2.5 µg/ml), significantly decreased forward scatter (10 µg/ml), significantly increased Fluo3 fluorescence (10 µg/ml), significantly increased ceramide abundance (10 µg/ml), significantly increased hemolysis (10 µg/ml), but significantly decreased DCFDA fluorescence (10 µg/ml). The effect of carnosic acid on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca²⁺. Conclusion: Carnosic acid triggers cell shrinkage and phospholipid scrambling of the human erythrocyte cell membrane, an effect paralleled by and/or in part due to Ca²⁺ entry and increased ceramide abundance.

D-glucose-induced alterations in the electrical parameters of human erythrocyte cell membrane

The alterations of the passive electrical parameters (the permittivity epsilon and the electrical conductivity sigma) of human erythrocyte cell membrane induced by the presence of glucose in the extracellular medium have been investigated by means of dielectric spectroscopy measurements. The membrane permittivity epsilon(s) and the permittivity epsilon(p) and electrical conductivity sigma(p) of the cytosol have been evaluated on the basis of a recent analytical model proposed by Prodan et al., 1983 [16], that takes into account the whole dielectric spectrum of a cell suspension, consisting of both the low-frequency alpha-dispersion and the high-frequency beta-dispersion. Our results show a marked increase of the membrane permittivity epsilon(s) close to a glucose concentration of 20mM. On the contrary, the electrical properties of the cytosol do not change appreciably. This finding strengthens the hypothesis that glucose interactions involve primarily the cell membrane and the mechanism of the transport is briefly discussed.

A study of porous structure of cellular membranes in human erythrocytes

Properties of cell membrane of human erythrocytes are studied using the mechanistic formalism of membrane transport developed earlier. We estimate that an erythrocyte with a membrane surface of 176 × 10 6 nm 2 has about 1900 water-permeable pores with cross-section areas ranging from 0.07 to 0.2 nm 2.

Effect of the shape of human erythrocytes on the evaluation of the passive electrical properties of the cell membrane

The possible influence of the cell shape on the derivation of the passive electrical parameters of a biological cell membrane is discussed in light of two different models which describe the cell as a shelled ellipsoidal particle and as a biconcave disk obtained by the revolution of the Cassini oval, respectively. Whereas within the first model, the Laplace equation can be solved analytically, in the second one a numerical algorithm based on the boundary element method has been employed. We have compared the results obtained by these two different models in the case of normal human erythrocyte cell membrane, using radiowave dielectric spectroscopy measurements. Our findings show that, although in principle the cell shape might deeply affect the evaluation of the passive electrical parameters of the cell membrane, in the case of the erythrocyte shape modelled by the Cassini curve, only small deviations are evidenced in comparison to the values derived, as usually done in the dielectric spectroscopy of biological cell suspensions, from an ellipsoidal model analysis. This result gives further support to the reliability of the data reported in the literature based on an ellipsoidal shape erythrocyte model.

Binding characterization of a putative cGMP transporter in the cell membrane of human erythrocytes.

Cyclic GMP is an intracellular signal molecule whose biological and pharmacological role is not well understood. Recent studies with human erythrocytes and other cell types (normal and transformed) have shown that the extrusion of cGMP is an ATP-dependent and saturable process. In this paper, we present our studies on binding of [3H]-cGMP to human erythrocyte ghost and its solubilized extracts. At 4 degrees C, an apparent dissociation constant of 0.15 microM was found in the samples. Maximum specific binding values in ghost and solubilized extracts were 9.0 pmol/mg of protein and 1.0 pmol/mg of protein, respectively. The low dissociation constant was confirmed by kinetic studies with a value of 0.16 microM. Specific [3H]-cGMP binding was inhibited by cAMP, cGMP, and cIMP with KD values of 0.22 microM, 0.09 microM, and 0.17 microM, respectively. Unlabeled cGMP and cIMP inhibited [3H]-cGMP binding completely whereas cAMP inhibited only 70%. The membrane-localized cGMP-binding protein discriminates between cyclic and noncyclic nucleotides, since GMP, IMP, and AMP were unable to displace [3H]-cGMP. A zwitterionic detergent, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate), was able to solubilize a protein with identical binding affinity. The results of this study show that erythrocyte ghosts possess a cGMP-binding protein which is not a kinase (due to a similar affinity for cAMP, cGMP, and cIMP) or phosphodiesterase (due to the inability of IBMX, 3-isobutyl-1-methylxanthine, to inhibit specific [3H]-cGMP binding). We hypothesize that this protein is the cell membrane cGMP transporter.

Cell membrane fluctuations are regulated by medium macroviscosity: evidence for a metabolic driving force.

Extracellular fluid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body fluids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mechanism of this phenomenon, we examined the effect of EFM on mechanical fluctuations of the cell membrane of human erythrocytes. The macroviscosity of the external medium was varied by adding to it various macromolecules [dextrans (70, 500, and 2,000 kDa), polyethylene glycol (20 kDa), and carboxymethyl-cellulose (100 kDa)], which differ in size, chemical nature, and in their capacity to increase fluid viscosity. The parameters of cell membrane fluctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, regardless of the cosolvent used to increase EFM. Because thermally driven membrane fluctuations cannot be damped by elevation of EFM, the existence of a metabolic driving force is suggested. This is supported by the finding that in ATP-depleted red blood cells elevation of EMF did not affect cell membrane fluctuations. This study demonstrates that (i) EFM is a regulator of membrane dynamics, providing a possible mechanism by which EFM affects cell membrane activities; and (ii) cell membrane fluctuations are driven by a metabolic driving force in addition to the thermal one.

Correlation between local cell membrane displacements and filterability of human red blood cells

Local mechanical fluctuations of the cell membrane of human erythrocytes were shown to involve MgATP- and Mg 2+-driven fast membrane displacements. We propose that these local bending deformations of the cell membrane are important for cell passage through capillaries. In order to verify this hypothesis, we examined cell membrane fluctuations and filterability of erythrocytes over a wide range of medium osmolalities (180–675 mosmol/kg H 2O). The results indicate the existence of a positive correlation between the amplitude of local cell membrane displacements and cell filterability. We suggest that the occurrence of metabolically driven membrane displacements on the side surface of the red blood cell diminishes its bending stiffness and enables it to fold more efficiently upon entrance into blood capillaries. Thus, local cell membrane displacements seem to play an important role in microcirculation.

Characteristics of human erythrocyte insulin binding sites.

Insulin and human erythrocyte cell membrane interactions were studied with respect to binding and dissociation. The per cent of specific binding of 125I-labeled insulin to erythrocytes was directly proportional to the cell concentration. The optimum pH for binding was 8.1. The initial binding rate was directly proportional to, and the steady state insulin binding was reversely proportional to, the incubation temperature. The per cent of specific binding of 125I-labeled insulin was 12.10 +/- 1.13 per cent (mean +/- SD)/4 X 10(9) cells (n = 10) at 0.8 ng/ml insulin. Native insulin competed with 125I-labeled insulin for binding and showed almost complete inhibition at 10(4) ng/ml. The Scatchard plots were upward concave. Maximum binding capacity was 230 binding sites per cell. The average affinity constant decreased as the per cent of fractional occupancy increased. Affinity constants for the empty and filled sites were 1.49 and 0.16 X 10(8) M-1 respectively. Bound insulin was displaced by native insulin. The dissociation rate by "dilution + native insulin" was higher than that by "dilution only". The dissociation rate was accelerated even by the physiological concentration of insulin and maximum at 100 ng/ml. It is concluded that human erythrocytes have insulin binding sites which are indistinguishable from insulin receptors on the target tissues for insulin.

Electrochemical properties of the cell membrane-electrolyte solution interface from microelectro-phoretic and contact potential difference measurements

We have tested two electrochemical methods: microelectrophoresis and contact potential difference measurements for their cooperation and in order to check if their results are complementary to one another in the in vitro studies of the cell membrane-electrolyte solution interface. Results of studies on cell membrane of human erythrocytes including the tetramethylammonium ion transport through the membrane, the La3+ cation adsorption, the dynamical change in the cell membrane structure in non-isotonic media, and the incubation time of erythrocytes in ACD solution indicate that the methods cooperate and that their results are complementary to one another.

Electrochemical properties of the cell membrane-electrolyte solution interface from microelectrophoretic and contact potential difference measurements

We have tested two electrochemical methods: microelectrophoresis and contact potential difference measurements for their cooperation and in order to check if their results are complementary to one another in the in vitro studies of the cell membrane-electrolyte solution interface. Results of studies on cell membrane of human erythrocytes including the tetramethylammonium ion transport through the membrane, the La 3+ cation adsorption, the dynamical change in the cell membrane structure in non-isotonic media, and the incubation time of erythrocytes in ACD solution indicate that the methods cooperate and that their results are complementary to one another.

Differential solubilization of human erythrocyte cell membrane proteins by maleic anhydride

A method is described to differentially solubilize the proteins of erythrocyte membranes using maleic anhydride. Based on this solubilization, the two groups of proteins appear to be distinct in their molecular organization within the membrane structure. From the operational criteria of peripheral and integral proteins of biological membranes, it is suggested that maleic anhydride by itself solubilizes mostly the peripheral proteins, while, in the presence of urea, it solubilizes almost all the proteins. It is suggested that maleic anhydride could be used to solubilize, fractionate and characterize the membrane proteins. Since maleyl groups can be removed from proteins, reconstitution of membrane structure from solubilized components could be studied.

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.